2017 Cardiovascular Research Day Abstract Book

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Recapitulating In Vivo Fibroblast Differentiation Using a Hydrogel Cell Culture System
Demetria Fischesser 1 • Onur Kanisicak, PhD 1 • Mario Perera 2 • Neil Ayres, PhD 2 • Jeff
D Molkentin, PhD 1
1Molecular Cardiovascular Biology, Cincinnati Children's Hospital • 2 Chemistry, University of
Cincinnati
Graduate Student
Cardiac fibroblasts are resident interstitial cells known to maintain the extracellular matrix (ECM)
under normal physiological conditions through the secretion of collagens and other adherent
proteins. During injury, these fibroblasts take on an activated, contractile state and deposit
additional ECM proteins to contribute to scar formation. While this response is initially beneficial to
prevent rupture and further injury, continuous scar development leads to fibrosis, pathological
ventricular remodeling and dysfunction, and eventual organ failure. Though much has already been
discovered about the importance of fibroblasts during development and injury response, there are
still many aspects of their functions that remain unclear. In order to decipher these fibroblast
functions, many in vitro systems have been used to easily manipulate fibroblasts while attempting
to mimic in vivo conditions. However, most of these systems have used plastic or gelatin-coated
plastic as the surface medium. These have proven to be much stiffer than physiological tissue and
provide a static system inhospitable to the dynamic changes of the activating fibroblast. In order to
mimic an adaptable in vivo environment for fibroblast culture, we have developed a new hydrogel
technology to understand fibroblast behavior and differentiation. This technology utilizes gelatin
norbornene and a thiol conjugated to a Reversible Addition-Fragmentation Chain Transfer (RAFT)
polymer which can dynamically mimic the stiffness of both healthy and fibrotic tissue, thus allowing
for seamless modeling of injury and disease in various tissues. Through morphology and
differentiation studies, we have found that there is a significant difference between fibroblasts
plated on plastic versus softer hydrogels which mimic in vivo conditions. Fibroblasts plated on
plastic and stiffer surfaces become very large and flat, and express markers of differentiation, such
as alpha-Smooth Muscle Actin (αSMA), within days of plating. On softer hydrogels, however, the
fibroblasts remain small and eventually develop long stellate appendages. They produce αSMA
much more slowly and at a much less robust level than on stiffer surfaces. The morphology and
differentiation pattern seen on softer hydrogels is highly comparable to fibroblasts observed in
vivo. We will use these initial findings to better understand the activation and differentiation of
fibroblasts and how, in injury, they first inhibit tissue damage and later enhance it. In the future, we
plan to manipulate this hydrogel system to intentionally change the stiffness of the surface on
which fibroblasts have already been plated to determine if we can mimic disease states as well as
reverse the fibroblast activation phenotype seen previously.
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